Session: 604. Molecular Pharmacology, Drug Resistance: Targeting Chemoresistant Cells and Niche
In this study, we investigated the anti-leukemic effects and molecular mechanisms of action of TGF-β neutralizing antibody 1D11 under hypoxic conditions. We further investigated the anti-leukemic efficacy of 1D11 combined with CXCR4 antagonist plerixafor in the in vivo leukemia models. AML cells (MV4;11 and U937) were propagated under 1% O2 for at least 14 days to assure their sustained proliferation and survival. Isotype control antibody 13C4 combined with ara-C induced no significant change in apoptosis or cell cycle progression. In MV4;11 cells cultured with 2ng/mL rhTGF-β1, 1D11 (10 μg/mL) induced only minimal apoptosis by itself, yet enhanced low-dose cytarabine (AraC, 0.5 μM) induced apoptosis. This effect was more prominent under hypoxia compared to normoxia (% of subG1 fraction, 21% O2: ara-C, 2.6 ± 0.2%, ara-C + 1D11, 10.8 ± 2.5%, p=0.03; 1% O2: ara-C, 11.3 ± 2.7%, AraC + 1D11, 21.4 ± 0.5%, p=0.001). 1D11 with ara-C abrogated rhTGFβ1-induced accumulation of cells in G0/G1 phase (21% O2; cont, 73.8 ± 4.1, rhTGFβ, 82.2 ± 3.2, rhTGFβ + AraC, 65.4 ± 2.5, rhTGFβ + AraC + 1D11, 50.3 ± 1.9, p=0.001: in 1% O2; cont, 71.8 ± 1.3, rhTGFβ, 85.4 ± 1.4, rhTGFβ + AraC, 79.3 ± 5.1, rhTGFβ + AraC + 1D11, 67.1 ± 4.0, p=0.03).
The anti-leukemic efficacy of 1D11 was next examined in an in vivo leukemia model. 1D11 administered at 5 mg/kg IP every other day in combination with ara-C (50 mg/kg IP weekly) decreased leukemia burden of nude mice injected with Baf3/ITD-luciferase leukemia cells (p=0.002). Administration of small molecule CXCR4 inhibitor plerixafor, which successfully diminished cell migration to CXCL12 in vitro, in combination with 1D11 decreased leukemia burden in vivo (p=0.05), and co-administration of ara-C, plerixafor and 1D11 was most effective (bioluminescence intensity, x107 photons/sec) control, 1.2 ± 0.2; ara-C, 0.94 ± 0.3; plerixafor + 1D11, 0.56 ± 0.1; plerixafor + 1D11 + ara-C, 0.23 ± 0.09, p=0.003).
We next examined the molecular mechanisms responsible for chemosensitization through blockade of TGFβ with 1D11. Treatment with rhTGF-β1 induced upregulation of p21 expression as well as pro-survival phosphorylation of Stat3 in MV4;11 and U937 cells, and these effects were abrogated by 1D11. Knock-down of Stat3 by siRNA increased apoptosis induction in U937 cells cultured in the presence of rhTGFβ1. Notably, 4-fold upregulation of the established TGFβ target, leukemia inhibitory factor (LIF) gene mRNA, was observed after rhTGF-β1 treatment and this was reversed by 1D11. These results indicate that 1D11 inhibits rhTGF-β1-induced autocrine stimulation of pro-survival LIF-JAK-Stat3 signal transduction pathway in AML cells.
In summary, blockade of TGF-β by 1D11, and abrogation of CXCL12/CXCR4 signaling may enhance the efficacy of chemotherapy against AML cells in the hypoxic BM microenvironment. These findings warrant further investigations in human clinical trials.
Disclosures: Konopleva: Genzyme: Research Funding.
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